Aspergillus in the Indoor Air of Critical Areas of a Tertiary Hospital in Brazil

Airborne Aspergillus spp. are critical pathogens that cause nosocomial infections in hospitals. Despite their importance, little is known about the distribution of Aspergillus species in the indoor air of hospitals in Brazil. We investigated Aspergillus spp. in the indoor air of critical areas in a tertiary hospital in Brazil. Air samples (n = 238) were collected from the intensive care unit (ICU), medical clinic unit (MCU), and urgency and emergency unit (UEU) using an air sampler (100 L/min). Of the 324 Aspergillus isolates, 322 were identified using phenotypic methods, and 37 were identified using DNA sequencing. Aspergillus spp. was grouped into five sections: Fumigati (29.3%), Nidulantes (27.8%), Nigri (27.5%), Flavi (11.7%), and Terrei (3.1%). The predominant species identified via sequencing were Aspergillus sydowii (n = 9), Aspergillus flavus (n = 7), and Aspergilus fumigatus (n = 6). The number of Aspergillus spp. and their sections varied according to the collection day. A. fumigatus was isolated more frequently during winter and in the ICU. This study is the first to demonstrate the diversity of airborne Aspergillus (saprophytic, allergenic, toxigenic, and potentially pathogenic) strains in a hospital located in the Midwest region of Brazil. It contributes to the knowledge of the diversity of cryptic species in the hospital environment.


Introduction
An environment contaminated with fungal spores plays an important role in the colonization of patients, and can precede infections such as invasive aspergillosis, a potentially lethal condition if not diagnosed and treated promptly [1][2][3][4].
Aspergillus species belonging to sections Nigri, Fumigati, and Flavi are most commonly described in hospital environments and clinical samples [2,3,5,7,[13][14][15].A. fumigatus is more prevalent than the other species, and an increase in antifungal-resistant isolates from clinical and environmental samples has been reported [3,7,11,12].Despite its significance, little is known about the distribution of airborne Aspergillus species in Brazilian hospitals [1].Aspergillus niger, A. fumigatus, and A. flavus were the most commonly found [14].
Approximately 62% of Aspergillus isolates in published Brazilian studies have not been identified at the species level [14].This study describes for the first time the different sections and species of Aspergillus in the indoor air of critical hospital areas in the state of Mato Grosso do Sul, Brazil, contributing to our knowledge of the biodiversity of these microorganisms in hospital environments.

Collection Procedure
Air samples were collected every 15 days in January, February, March, August, and December 2021 between 2:00 pm and 4:00 pm from Campo Grande, the capital of Mato Grosso do Sul State, in the Midwest region of Brazil.University Hospital Maria Aparecida Pedrossian (UHMAP) is a public tertiary teaching hospital.Geographically, Campo Grande is located near the borders of Brazil, Paraguay, and Bolivia.The tropical climate is characterized by dry winters and humid summers, with heavy rainfall and high temperatures.Humidity, temperature, and rainfall information were obtained from Mato Grosso do Sul Weather and Climate Monitoring Center website (https://www.cemtec.ms.gov.br/bancodedados/2021-2), accessed on 21 July 2022 and corresponded to the days and times of collection.
Air collection was performed in the intensive care unit (ICU), in the medical clinic unit (MCU), and in the urgency and emergency unit (UEU).The MCU and the UEU do not have air conditioning.The ICU is equipped with wall air conditioners, which are periodically cleaned and maintained by a specialized company.None of the three units has a high efficiency air filtration system.Floors and surfaces are cleaned daily.
Of each hospitalization unit, nine air samples/day were collected using an air impactor (MiniCapt Microbial Air Sampler, Particle Measuring Systems, Boulder, CO, USA) adjusted to a volume of 100 L/min per 1 min on a plate containing Dichloran Rose-Bengal Chloramphenicol (DRBC) agar (Kasvi, Conda Laboratories, Madrid, Spain).Immediately after collection, petri dishes containing air samples were packed in individual plastic bags and transported to the laboratory in a closed box.The fungal colony plates were processed in a biological safety cabin to avoid possible sample contamination.The plates were then incubated at 30 • C for 72 h.In this study, fungal load corresponds to the number of filamentous fungi colony-forming units (CFU/m 3 ) of a Petri dish.

Fungal Identification
The collection sites were positioned 1.5 m above the ground and air was collected at the same points determined during the first sampling.
Aspergillus isolates (n = 324) were identified at the section level by analyzing their macroscopic and microscopic characteristics [16].Owing to financial constraints, only 37 isolates were randomly selected for molecular identification using polymerase chain reaction (PCR) and sequencing.Briefly, genomic DNA was extracted using a Biogene kit (Bioclin, Belo Horizonte, Brazil), following the manufacturer's instructions.PCR was performed as previously described [17,18].The isolated DNA fragments were amplified using primers targeting the β-tubulin and calmodulin regions (Table 1).The PCR products were purified using the ExoSap-IT Express kit (Applied Biosystes, Foster City, CA, USA) and sequenced using Bt2a/Bt2b primers in an ABI 3730xl System service provided by Macrogen facility (Seoul, South Korea).The sequences were checked for quality, and the forward and reverse sequences were concatenated using Geneious software (Geneious 7.1.3,Biomatters Ltd., Auckland, New Zealand).For species identification, nucleotide sequence comparisons were performed against the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST; GenBank accession OR758882-OR758901.

Statistical Analyses
ANOVA was used to evaluate whether there was a significant difference in the number of Aspergillus spp.collected per plate across collection days.The Pearson chi-square or the Fisher exact test (in cases where one of the cells had an expected value of ≤5) was applied to determine whether there was a difference in the type of Aspergillus collected from different hospital units and between seasons.
A Pearson correlation analysis was conducted to assess the influence of independent variables (humidity, temperature, and rainfall) on the dependent variable, total Aspergillus spp.The dataset comprises observations collected on different dates during the study period.Correlation coefficients (r) were used to measure the strength and direction of the relationships between independent and dependent variables.The p values were examined to determine the statistical significance of each correlation.Statistical analyses were performed using R (version 4.1.1)at the RStudio interface [21].

Results
Between January and December 2021, in the 238 air samples collected from the intensive care unit (ICU), Medical Clinic Unit (MCU), and the urgency and emergency unit (UEU), we observed the growth of 4300 filamentous fungi colonies.

Number of Filamentous Fungi Colony-Forming Units (CFU)
A total of 4295 filamentous fungi CFU were isolated from indoor air of UHMAP critical areas.The average number of filamentous fungal colonies isolated was 224 CFU/m 3 and the average number per air sample ranged from 7.4 to 42.4 CFU, with variations depending on the day of collection.Table 2 shows the filamentous fungal load in hospital air according to the collection date.A significant difference was found between the fungal load and the day of collection (p < 0.001).
The overall average number of Aspergillus spp.isolated per air sample ranged from 17 to 30 CFU/m 3 , depending on the day of collection.The CFU of Aspergillus colonies varied between the days of collection (p = 0.02).The daily distribution of Aspergillus sections is presented in Table 3.

Aspergillus Section Per Hospitalization Unit
Analysis of the number of Aspergillus colonies per hospitalization unit revealed that the ICU had the highest load (157; 48.5%) of Aspergillus colonies dispersed in the air.Fumigati was the most frequent section of the ICU (36.3%, 57/157).Aspergillus from the Nidulantes section (46/138) was more prevalent in the MCU, followed by that in the Nigri section (41/138).Figure 1 shows the distribution of Aspergillus spp.sections per hospital unit.

Aspergillus Load According to Section and Season
The variation in Aspergillus load according to section and season was significant (p = 0.001).Unfortunately, it was not possible to collect data during the autumn.Table 4 shows that Aspergillus species from the Fumigati section were more frequent in the winter (64, 67.4%).In contrast, species from the Nigri section (71; 79.8%) and Nidulantes (49; 54.4%) were isolated during the summer.
figfig:jof-3001606-f002 shows the Aspergillus spp load according to humidity, temperature, and rainfall on the collection dates.In winter, during periods of lower rainfall, we observed a larger load of Aspergillus in ambient air.Pearson correlation analysis indicated that humidity, temperature, and rainfall were not significantly correlated with the presence of Aspergillus spp. in indoor air.

Aspergillus Load According to Section and Season
The variation in Aspergillus load according to section and season was significant (p = 0.001).Unfortunately, it was not possible to collect data during the autumn.Table 4 shows that Aspergillus species from the Fumigati section were more frequent in the winter (64, 67.4%).In contrast, species from the Nigri section (71; 79.8%) and Nidulantes (49; 54.4%) were isolated during the summer.Figure 2 shows the Aspergillus spp load according to humidity, temperature, and rainfall on the collection dates.In winter, during periods of lower rainfall, we observed a larger load of Aspergillus in ambient air.Pearson correlation analysis indicated that humidity, temperature, and rainfall were not significantly correlated with the presence of Aspergillus spp. in indoor air.

Discussion
The results showed saprophytic, allergenic, toxigenic, and potentially pathogenic Aspergillus strains in the air of critical areas of the studied hospital.This study provides for the first time the molecular identification of Aspergillus species from the indoor air of critical hospital areas in the Midwest region of Brazil.Globally, few studies have evaluated the microbial quality of indoor air in hospitals (or hospital environments).
Despite their relevance, there are no specific regulations or guidelines that specify limits for fungi in the indoor air of hospitals [22].According to the standards of the Brazilian Ministry of Health and the National Health Surveillance Agency, the Maximum Recommended Value for fungal contamination of indoor air is 750 CFU/m 3 in indoor air [23].Although the average number of filamentous fungal colonies found in environmental air in our study was within the limits established by Brazilian law, this value (224 CFU/m 3 ) was higher than those previously described in other Brazilian hospitals.Pantoja et al. (2012) [24] described that, in air collected from the ICUs of three hospitals in northeastern Brazil, the number of fungal spores ranged from 43.75 to 73.67 CFU/m 3 .In the indoor air of three ICUs of two university hospitals in southern Brazil, the median overall fungal concentrations for ICU 1, 2, and 3 were 109.5, 134.3, and 55.7 CFUm 3 , respectively [1].
In this study, the source of air contamination by Aspergillus spores is uncertain.External and internal environmental factors and other predisposing conditions may be involved.In the medical clinic unit (MCU), and in the emergency unit (UEU) where there is no air conditioning and the windows are open, there is the possibility that the indoor Aspergillus can, in part, originate from an outdoor environment via the airflow.In these hospital units, the movement of people can also favor the dispersion of fungal conidia that grow on surfaces [25].In the ICU, where the movement of people is quite restricted, and there is no direct access to the external environment, it is believed that the aerocontamination by Aspergillus is mainly due to internal sources.In enclosed environments, without a high efficiency air filtration system or with an improperly maintained air system, bacteria and fungi can spread and survive in the environment [26,27].
Our results are particularly relevant for hospital units to which immunosuppressed patients and those with chronic diseases are admitted, such as intensive care units and medical clinics.The results revealed that the average number of filamentous fungal colonies (including Aspergillus spp.) per plate varied significantly, depending on the day of collection (p < 0.05).Previous studies have shown that in hospitals, where many spores can circulate in the air, the risks of both infection and hospital infection outbreaks increase significantly [1,26,28].
The findings of this study prompted the Hospital Infection Control Commission to act, as fungi are recognized as indicators of indoor air quality.Support from senior hospital management was sought to implement routine air analysis.Additionally, meetings were held with the engineering team to discuss interventions for enhanced environmental control, such as installing barriers to prevent air infiltration from construction or renovation activities, and regular maintenance of air conditioning equipment to reduce the amount of filamentous fungal spores.
Other measures to minimize the number of filamentous fungal spores in the internal air, include vacuum cleaning, restricting the entry and propagation of microorganisms in the internal environments, and using air cleaning devices.To control air quality more effectively and reduce fungal colony counts, it may be necessary to install heating, ventilation, and air conditioning (HVAC) and high efficiency particulate air (HEPA) filtering systems [13,22].
One of the limitations of this study is that for logistical reasons, outdoor air samples could not be obtained, which could have provided more accurate data on the distribution of these fungi in the environment.Sample collections were consistently conducted simultaneously prior to routine cleaning of the site to minimize potential sources of interference.The higher number of colonies observed on specific days may have been influenced by various internal or external factors, such as the movement of patients, healthcare teams, visitors, and employees, as well as minor renovations within the hospital [13,29,30].
As observed in the present study, filamentous fungi present in the internal air of hospitals may aggravate asthma and allergic rhinitis and cause serious diseases in susceptible individuals [2,4,9,[31][32][33][34].
The analysis of the microbial load of fungi in indoor air is challenging.In this study, on the last day of collection (14 December 2021), the number of filamentous fungi was higher in almost all sectors, indicating that the air in the hospital had a high load of filamentous fungi.Future studies should aim to correlate the genotypes of clinical and environmental isolates collected during the same period to determine the impact of high concentrations of airborne spores dispersed in the air.
Aspergillus from the Fumigati section is considered the main cause of invasive aspergillosis and was the most prevalent agent in this study.Unlike what has been described in other countries and other Brazilian regions, in the studied hospital, Aspergillus from the Nidulantes section was the second most frequent, with a similar quantity to species from the Nigri section, which tends to be the second most frequent section in the indoor air of hospitals [12,13,15].Aspergillus belonging to the Fumigati and Nigri sections was prevalent in the indoor air of UHMAP, corroborating studies in Asia [13], Europe [12,15], Africa [25], and the American continent [1].In Brazil, these fungi have been described in hospital environments in the Northeast [24,27,35,36], Midwest [37], Southeast [38], and South [1,39] regions.Most Brazilian studies have not identified Aspergillus at the species level, limiting our knowledge of the most prevalent species in hospitals in Brazil [14,26].
In the present study, significant differences were observed in the load of sections of Aspergillus spp., according to the sampling date.In the first quarter of 2021, isolates from the Nigri and Nidulantes sections were predominant.This period was characterized by heavy rainfall.High humidity may have contributed to the many Aspergillus isolates found in these sections [8].Aspergillus species from the Nigri and Nidulantes sections are routinely recovered from hospital environments and can cause severe diseases [8,31,40] Aspergillus from the Fumigati section was the most common species isolated from ICU patients (36.3%).This finding is crucial because immunocompromised patients are vulnerable to fatal respiratory infections caused by A. fumigatus [4,9,41].Species from the Flavi section were also frequently found in the ICU.According to a recent study, A. flavus is the second most common Aspergillus species isolated from invasive aspergillosis patients [10].The presence of Aspergillus isolates from the Fumigati and Flavi sections in the critical care units found in our study highlights the urgent need to implement measures to improve air quality, not only in the units studied but also in other hospital units.
Previous studies have shown that temperature and relative humidity positively correlate with the growth and reproduction of fungi in hospital environments [42][43][44].On December 14, considered summer in Brazil, the largest load of filamentous fungi found may be due to high relative humidity, which on this day was greater than 80%.However, in our study, the environmental parameters evaluated (humidity, temperature, and rainfall) did not significantly influence the load of Aspergillus spp. in the internal air of the hospital.Similar to our results, Cho et al., 2018 [13] did not find a statistical correlation between Aspergillus sections in the internal air and temperature.This result may be related to the remarkable ability of some species, such as A. fumigatus, to adapt to the changing climate [45].In fact, little is known about the variations between Aspergillus strains and their growth at different temperatures and how their geographical origin affects such variations [45].Further studies are needed to verify how these environmental parameters correlate with the presence of different Aspergillus species.
The study hospital is located in a tropical climate characterized by dry winters and humid summers with heavy rain and high temperatures.Although we did not observe a correlation between the number of Aspergillus isolates in the air and the temperature, it was observed that during the winter, when there was less rain, more CFU of Aspergillus spp.were isolated on average per plate.One of the limitations of this study was the inability to collect air samples during the autumn owing to the COVID-19 pandemic.The impossibility of collecting samples in autumn influenced the total number of filamentous fungi, the number of Aspergillus (sections and species) isolated, and other results related to seasonality.However, we could not evaluate the impact of the lack of such data in the present study.The reason some Aspergillus species are more prevalent in summer and winter is not well established.Seasonal fluctuations in airborne fungal concentrations are influenced by various factors, with geoclimatic conditions being a significant contributor [13,42,45].
Statistical analyses revealed differences in Aspergillus sections and sampling periods in terms of fungal cell counts, indicating that seasonality might be related to an increase in certain species of the genus.This investigation revealed that species from the Fumigati section were isolated in greater numbers during periods of drought, particularly in the winter.This trend is consistent with the characteristics of A. fumigatus, which thrives under such conditions [46]. A. fumigatus spores are highly conducive to air dispersion owing to their small size and significantly higher hydrophobicity than those of other Aspergillus species, allowing them to remain airborne for longer periods [6].A study conducted in the USA reported that the incidence of aspergillosis was associated with seasonal periods of low precipitation and high temperatures.According to the authors, hot and dry weather conditions facilitate greater dispersal of hydrophobic conidia, which are critical factors in the transmission of aspergillosis [44].
In this study, Aspergillus isolates were initially identified based on their macro-and microscopic characteristics, and almost all were classified into sections.However, phenotypic identification of Aspergillus fungi is challenging and has certain limitations.This is because these fungi share many species similarities and can exhibit morphological variations dependent on culture medium or incubation temperature.Moreover, phenotypic identification requires analysts to have a certain level of experience [16].
The results of this study help fill a gap in the knowledge of Aspergillus species present in the internal air of hospitals in Brazil.Figure 3 shows the distribution of Aspergillus species identified in the indoor air of hospitals in only three of the five regions of Brazil: Northeast [27,36], Midwest [37], and Southeast [38,47].Despite their high sensitivity and specificity, molecular techniques are of moderate complexity, require specialized technical knowledge, and have high capital costs [48], which makes it challenging to implement the routine of hospital laboratories from low income regions.
Molecular identification revealed cryptic species (which are difficult to distinguish by morphological identification and exhibit distinctive molecular characteristics), including A. sydowii (n = 9), A. tubingensis (n = 4), A. pseudotamarii (n = 2), A. luchuensis (n = 1), A. uvarum (n = 1), A. japonicus (n = 1), and A. pseudocaelatus (n = 1) in the hospital's indoor air. A. sydowii (Nidulantes section) and A. tubingensis (Nigri section) are among the most common Aspergillus cryptic species in human pathology [3,49,50].A. sydowii is known to cause allergies and aggravate asthma, and is rarely involved in endophthalmitis, aspergilloma, and invasive pulmonary aspergillosis [34,50].A. tubingensis is commonly found in indoor air and has been described as an agent of otitis, keratitis, and lower respiratory tract infections in hospitalized patients [3,7].Nosocomial infections caused by these agents are difficult to treat because of their reduced sensitivity to amphotericin B and intrinsic resistance to azoles [3,10,49,50].A. luchensis, another important member of the Aspergillus species belonging to the Nigri section, can cause invasive aspergillosis [51]. A. pseudotamarii, an aflatoxigenic member of the Aspergillus section Flavi, has been identified as a mycotic keratitis agent [52]. A. japonicus is one of the main allergenic fungi in India and is considered an agent of pulmonary aspergillosis in patients with COVID-19 [3,53,54].A. pseudocaelatus is a toxigenic fungus that produces aflatoxins in various crops [55].Improvements in microbiology laboratories and the widespread use of molecular diagnostic tools will facilitate more precise species descriptions [56].microscopic characteristics, and almost all were classified into sections.However, phenotypic identification of Aspergillus fungi is challenging and has certain limitations.This is because these fungi share many species similarities and can exhibit morphological variations dependent on culture medium or incubation temperature.Moreover, phenotypic identification requires analysts to have a certain level of experience [16].
The results of this study help fill a gap in the knowledge of Aspergillus species present in the internal air of hospitals in Brazil.Figure 3 shows the distribution of Aspergillus species identified in the indoor air of hospitals in only three of the five regions of Brazil: Northeast [27,36], Midwest [37], and Southeast [38,47].Despite their high sensitivity and specificity, molecular techniques are of moderate complexity, require specialized technical knowledge, and have high capital costs [48], which makes it challenging to implement the routine of hospital laboratories from low income regions.Molecular identification revealed cryptic species (which are difficult to distinguish by morphological identification and exhibit distinctive molecular characteristics), including A. sydowii (n = 9), A. tubingensis (n = 4), A. pseudotamarii (n = 2), A. luchuensis (n = 1), A. uvarum (n = 1), A. japonicus (n = 1), and A. pseudocaelatus (n = 1) in the hospital's indoor air. A. sydowii (Nidulantes section) and A. tubingensis (Nigri section) are among the most common Aspergillus cryptic species in human pathology [3,49,50].A. sydowii is known to cause allergies and aggravate asthma, and is rarely involved in endophthalmitis, aspergilloma, and invasive pulmonary aspergillosis [34,50].A. tubingensis is commonly found in indoor air and has been described as an agent of otitis, keratitis, and lower respiratory tract infections in hospitalized patients [3,7].Nosocomial infections caused by these agents are difficult to treat because of their reduced sensitivity to amphotericin B and intrinsic resistance to azoles [3,10,49,50].A. luchensis, another important member of the Aspergillus species belonging to the Nigri section, can cause invasive aspergillosis [51]. A. pseudotamarii, an aflatoxigenic member of the Aspergillus section Flavi, has been identified as a mycotic keratitis agent [52]. A. japonicus is one of the main allergenic fungi in India and is considered an agent of pulmonary aspergillosis in patients with COVID-

Conclusions
Aspergillus fungi, including species from the Fumigati, Nidulantes, Nigri, and Flavi sections, were found in the indoor air of a tertiary referral hospital for infectious diseases in the mid-western region of Brazil.
Isolates from the Fumigati section were more prevalent during the winter months; however, additional research is needed to establish the seasonality of these fungi.
The presence of A. fumigatus in intensive care units increases the risk of aspergillosis caused by these pathogens, and emphasizes the need for measures to improve air quality in the hospital environment.

Figure 1 .
Figure 1.Aspergillus distribution according to section and collection site.UHMAP Campo Grande-MS, January-December 2021.

Figure 1 .
Figure 1.Aspergillus distribution according to section and collection site.UHMAP Campo Grande-MS, January-December 2021.

Table 4 . 13 Figure 2 .Figure 2 .
Figure 2. Aspergillus spp.load according to humidity, temperature, and rainfall on the date of collection.Campo Grande-MS, 2021.Note: Humidity (%) represents the percentage of humidity recorded on each collection date.Temperature (°C): Indicates the temperature in degrees Celsius observed on each collection date.Rainfall (mm): The amount of precipitation in millimeters measured on each collection date.Aspergillus: Presents the load of Aspergillus spp. on each collection date.Explanatory Note: This grouped bar chart illustrates the climatic variations and load of Aspergillus on nine different collection dates throughout 2021.Each group of bars represents measurements of humidity, temperature, and precipitation (rainfall) associated with a specific date.The above legend describes the climatic variables represented by distinct colors, and the gold color indicates the load of Aspergillus.The heights of the bars represent the values of each variable, whereas the secondary axis on the right displays the load of Aspergillus.Source: Mato Grosso do Sul State Weather and Climate Monitoring Center (CEMTEC/SEMAGRO).The Pearson correlation Figure 2. Aspergillus spp.load according to humidity, temperature, and rainfall on the date of collection.Campo Grande-MS, 2021.Note: Humidity (%) represents the percentage of humidity recorded on each collection date.Temperature ( • C): Indicates the temperature in degrees Celsius observed on each collection date.Rainfall (mm): The amount of precipitation in millimeters measured

Figure 3 .
Figure 3. Distribution of species of Aspergillus identified in indoor air of Brazilian hospitals.

Figure 3 .
Figure 3. Distribution of species of Aspergillus identified in indoor air of Brazilian hospitals.

Table 1 .
Primer used in DNA amplification and sequencing for molecular identification of Aspergillus species.

Table 3 .
Distribution of 324 Aspergillus species grouped by section, per day of collection.UHMAP-Campo Grande-MS, 2021.

Table 4 .
Aspergillus sections scattered in the indoor air during the UHMAP season (Campo Grande-MS, 2021).